Serotonin (5-hydroxytryptamine, 5-HT) is an evolutionary ancient biochemical, widespread throughout the animal and plant kingdoms. In mammals, serotonin acts as a neurotransmitter within the central and peripheral nervous systems (CNS, PNS) and as a local hormone in various other non-neuronal tissues, including the gastrointestinal tract, the cardiovascular system and immune cells. This functional duality of the serotonin system is typical for all vertebrates.
Within mammalian organisms only a few cell types synthesize serotonin, indicated by the expression of tryptophan hydroxylase (TPH) which is the initial and rate-limiting enzyme in the biosynthesis of serotonin. The multiplicity of serotonin actions is linked to many complex physiological and pathological functions. In mammals, about 70-90% of the total serotonin resides in the gastrointestinal tract, assisting digestive activities. There, it is mainly produced by enterochromaffin cells (EC) and by neurons of the enteric nervous system (ENS). Both cell types release serotonin upon mechanical or chemical stimuli, to induce contraction of smooth muscle cells and to regulate intestinal motility, secretion and intestinal blood flow. Serotonin from EC also enters the circulation and is taken up by thrombocytes and stored in specific vesicles. Platelet-derived serotonin plays a role in liver regeneration and primary haemostasis after vessel injury. Peripheral serotonin is also known to be involved in pulmonary hypertension, cardiac function, cardiac morphogenesis, ontogenesis, mammary gland plasticity, cancer, T-cell-mediated immune response and insulin secretion from pancreatic β-cells. The highest concentration of peripheral serotonin is found in the pineal gland, where it serves as precursor molecule for the biosynthesis of melatonin, a neuronal hormone involved in many physiological processes like thermoregulation and sleep.
Because of its hydrophilic properties, serotonin is not able to penetrate the blood-brain barrier (BBB). Therefore it needs to be synthesized in the brain, by serotonergic raphe neurons of the brainstem.
Central serotonin is important for the brain development. Furthermore, it is partaking in the regulation of sleep, body temperature, respiratory drive, motor control, CNS vascular tone, pain sensation and nociception. In addition, serotonin affects nearly all behavioural patterns, including memory, general mood, stress response, aggression, fear, appetite, addiction as well as maternal and sexual behaviour. An imbalance in the serotonin system has been implicated in a multitude of neuropsychiatric diseases.
The biosynthesis of serotonin is a highly regulated two-step process, starting with the essential amino acid L-tryptophan (Trp), cf. scheme below. The first and rate-limiting step comprises the hydroxylation of Trp to 5-hydroxytryptophan (5-HTP). This reaction is carried out by the enzyme tryptophan hydroxylase (TPH) and requires Fe2+ ions as a cofactor and molecular oxygen (O2) and tetrahydrobiopterin (BH4) as co-substrates. Two isoforms of TPH (TPH1 and TPH2) exist, reflecting the functional duality of serotonin on the biochemical level. Secondly, 5-HTP is immediately decarboxylated to 5-hydroxytryptamine (5 HT) by the ubiquitously expressed aromatic amino acid decarboxylase (AAAD).

Biosynthesis of Serotonin (5-HT).
TPH1/2: Tryptophan hydroxylase 1 and 2, AAAD: Aromatic amino acid decarboxylase.
TPH1 and TPH2 proteins in vertebrates are highly homologous, sharing an overall 70% amino acid sequence identity in humans, but differ in their kinetic properties and, more remarkably, in their tissue distribution. Further studies of mRNA and protein levels in rodent and human tissues confirmed TPH2 to be the central isoform, predominantly expressed in raphe neurons of the brainstem and in peripheral myenteric neurons in the gut, while it is absent in peripheral organs, such as lung, heart, kidney or liver. On the other hand, TPH1 is mainly found in the gastrointestinal system as well as in the pineal gland, where it produces serotonin serving as a precursor molecule for melatonin biosynthesis.
The disability of serotonin to cross the BBB enforces the dualistic character of the serotonin system by creating two physiologically separated serotonin pools in the body. In fact, both serotonin systems are defined by the TPH1 and TPH2 isoforms and characterized by distinct physiological functions and independent regulatory mechanisms. Consequently, both systems can be targeted in an autonomous fashion to pharmacologically or genetically manipulate central and peripheral serotonin functions.
The catalytic domain of TPH is highly conserved and incorporates all of the residues required for enzyme activity and substrate binding. Data from X-ray structures of the catalytic domain helped to establish the structure of the active site and to reveal amino acid residues involved in substrate and cofactor binding. The carboxylate group of Trp interacts with Arg257 and Asp269, while the Trp side chain is held in a hydrophobic pocket formed by Pro268, His272, Phe313 and Phe318. The co-substrate BH4 interacts with Phe241 and Glu273. Ligands to the non-heme iron (Fe2+) are His272, His277 and Glu317 are referred to as the 2-His-1-carboxylate facial triad. The general catalytic mechanism involves the iron-mediated incorporation of one atom of molecular oxygen into both the Trp substrate and the reducing co-substrate BH4, yielding a hydroxylated product. This reaction is subdivided in three different steps, starting with the formation of an iron-peroxypterin and followed by its decay to a reactive intermediate and subsequent Trp hydroxylation via electrophilic aromatic substitution.
A variety of diseases are associated with a dysregulation of serotonin synthesis and metabolism. One example is carcinoid syndrome, a collection of symptoms resulting from an excessive release of hormones by carcinoid tumors. Carcinoid tumors develop from enterochromaffin cells, which produce serotonin, dopamine, tachykinins, and other substances that can have profound effects on the circulatory system, the gastrointestinal tract, and the lungs. Other serotonin-related cancer diseases comprise cholangiocarcinoma and neuroendocrine (N E) cancers, such as carcinoids and pancreatic endocrine tumors, prostate cancer.
A number of documents addresses compounds capable of influencing the serotonin level, in particular by inhibiting TPH (e.g. WO 2011/100285 A1, US 2009/0048280 A, WO 2010/003997 A1, US 2009/0088447 A1). The structures disclosed in WO 2011/100285 neither comprise a xanthine moiety nor a benzimidazolyl group.
However, because serotonin targets multiple receptors and is involved in so many biochemical processes, drugs that interfere with serotonin signalling are often attended by adverse effects. Thus, a need exists for new methods of affecting serotonin levels.
Lei Zhang et al. (“Discovery of Novel Vascular Endothelial Growth Factor Receptor 2 Inhibitors: A Virtual Screening Approach”; Chem. & Biol. Drug Des. 80 (2012), p. 893-901) disclose a benzimidazolyl xanthine derivative with potential use as inhibitor for vascular endothelial growth factor 2.
Henrik Frandsen et al. (“N-acetyltransferase-dependent activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine”; Carcinogenetics 21, 6 (2000), p. 1197-1203) describe a hydroxylated derivative of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as a urinary biomarker for PhIP. PhIP is known to be a mutagenic and carcinogenic heterocyclic amine formed during frying of meat.